Abstract: This study explored the effects of millet bran pretreatment methods and fresh level on the extraction rate of millet bran oil. The ultrasonic assist technology was used, and the optimal extraction solvent was selected. The extraction process of millet bran oil was optimized through single-factor experiments and response surface analysis. The main physical and chemical properties of millet bran oil were determined, and the fatty acid composition was analyzed. The results showed that fresh millet bran need be selected and treated with infrared heating at 121 ℃ for 15 min. Anhydrous ethanol could be used as the best extraction solvent. The optimized extraction conditions were the extraction time 2 h, the material to solvent ratio 1:6.5 g/mL, and the extraction temperature 56 ℃. Under this condition, the extraction rate of millet bran oil was 78.57%. The order of various factors on the extraction rate of millet bran oil was: Material to solvent ratio > extraction temperature > extraction time. The acid value and peroxide value of millet bran oil extracted under this conditions were in line with the National Food Safety Standard (GB2716-2018) indicators for rice bran oil. The main fatty acid components of millet bran oil contained palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. The content of unsaturated fatty acid accounted for 90.58% of the total fatty acid content, of which the linoleic acid content was as high as 72.31%. The ultrasonic-assisted extraction process of millet bran oil with anhydrous ethanol proposed in this study was feasible and could provide important theoretical support for the further development and utilization of millet bran.